The specific aims of this project are to utilize antibodies made against specific bovine bone matrix proteins in order 1) to characterize fully the optimal culture conditions required for the synthesis and secretion of each of the proteins; 2) to correlate these culture conditions to those which will support maximal alkaline phosphatase activity; 3) to compare ultrastructurally the subcellular localization of these proteins in presecretory vesicles. Fetal bovine calvarial bone cells will be isolated utilizing collagenase treatment and grown in media containing 2-20 percent fetal calf serum. The culture conditions will be modified by addition of ascorbic acid, reduced glutathione or ITS (insulin, transferrin, selenium). Optimal culture conditions will be evalulated utilizing the following criteria: 1) examination of growing cultures microscopically to assess general appearance and growth characteristics; 2) application of the fluorescein-conjugated double antibody technique to cells to survey microscopically for the presence of matrix proteins and to semiquantitate their levels; 3) collection, concentration and immunoprecipitation assay (Elisa) on used culture media to identify bone matrix proteins; 4) utilization of the radioactive tracers 35SO4 or 35S-methionine to perform protein synthesis experiments by incubating cells in cell-free media for short-term experiments (pulse, continuous or pulse-chase labeling) or in modified media for long-term studies. The bone cells and media each will be analyzed with a combination of electrophoresis and autoradiography for specific bone proteins. In alkaline phosphatase experiments, both histochemical and analytical measurements will be compared and correlated to those conditions which support synthesis and secretion of each of the bone matrix proteins. For ultrastructural studies, cells will be grown in culture dishes with embeddable plastic inserts, fixed 15-20 minutes in 4 percent paraformaldehyde containing protease inhibitors or in 0.5 percent glutaraldehyde and 3 percent paraformaldehyde, the cell membranes rendered permeable with detergent and processed for ultrastructural immunocytochemistry using the peroxidase-antiperoxidase technique. The results of this project will provide the parameters for growth of bovine bone cells so that the effects of bone seeking hormones can be assessed and applied to the successful treatment of metabolic and genetic bone disease.